Tricyclic compounds having cytostatic and/or cytotoxic activity and methods of use thereof

ABSTRACT

The present invention provides tricyclic compounds having cytostatic and cytotoxic activity in a single molecule having receptor tyrosine kinase(s), dihydrofolate reductase, thymidylate synthase and/or dihydroorotate dehydrogenase inhibitory activity, which are useful as anti-angiogenic and anti-tumor agents. Also provided are methods of utilizing these inhibitors to treat tumor cells and other proliferative diseases and disorders.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to tricyclic heteroaromatic compounds andtheir methods of use and, more particularly, to tricyclic heteroaromaticcompounds that inhibit receptor tyrosine kinase(s), dihydrofolatereductase, thymidylate synthase and/or dihydroorotate dehydrogenaseactivity so as to exert cytostatic and cytotoxic action on tumor cellsand other proliferative diseases and disorders.

2. Description of the Prior Art

The formation of new blood vessels from existing vasculature is termedangiogenesis. Angiogenesis plays a crucial role in the growth andmetastasis of solid tumors. Solid tumors require angiogenesis to growbeyond 1-2 mm in diameter and metastasis requires the presence of bloodvessels to allow access to the circulation and to form tumors at distalsites to the primary tumor. Angiogenesis and metastasis contribute tothe poor prognosis in patients with angiogenic solid tumors. Thus,agents that inhibit the angiogenic process have afforded new paradigmsfor the treatment of tumors.

Angiogenesis primarily is a receptor-mediated process by growth factorsthat cause signal transduction, for the most part, by receptor tyrosinekinases (RTKs). RTKs consist of families of growth factor receptors suchas vascular endothelial growth factor receptor (VEGFR), epidermal growthfactor receptor (EGFR), platelet-derived growth factor receptor (PDGFR)and fibroblast growth factor receptor (FGFR). Aberrant expression oroverexpression of EGFR and PDGFR, both of which are directly orindirectly involved in angiogenesis, have been implicated in thedevelopment, progression and aggressiveness of a variety of solidtumors. These include head and neck cancers (Shin, D. M. et al., CancerRes., 54:3153-3159, 1994), non-small cell lung cancer (Tateishi, M. etal., Cancer Res., 50:7077-7080, 1990; Gorgoulis, V. et al., AnticancerRes., 12:1183-1187, 1992), glial tumors (Fleming, T. P. et al., CancerRes., 52:4550-4553, 1992) and glioblastomas (Fleming, T. P. et al.,Cancer Res., 52:4550-4553, 1992; Maxwell, M. et al., J. Clin. Invest.,86:131-140, 1990; Hermanson, M. et al., Cancer Res., 52:3213-3229,1992).

Because RTKs are present in endothelial cells (VEGFR, PDGFR), tumorcells (FGFR, PDGFR) and pericytes/smooth muscle cells (FGFR, PDGFR),inhibition of more than one RTK may provide synergistic inhibitoryeffects against solid tumors. Thus, RTKs are attractive targets forcancer chemotherapeutic agents.

The importance of multiple RTK inhibition in angiogenesis is wellrecognized for the treatment of diseases such as cancer and maculardegeneration, with multiple compounds in clinical use and severalcurrently in various phases of clinical trials. These compounds,however, only are cytostatic, stopping the growth of tumors by blockingthe angiogenesis pathway, and thus depriving tumors of the nutritionthey need to grow. Hence, the antiangiogenic effect of these compoundsdoes not kill tumor cells. To kill tumor cells, an additional cytotoxiceffect is necessary. This cytotoxic effect can be provided by existingcancer chemotherapeutic agents. Thus, a variety of RTK inhibitors thatare antiangiogenic and cytostatic have been combined with existingcancer chemotherapeutic agents that are cytotoxic, such as dihydrofolatereductase (DHFR) and thymidylate synthase (TS) inhibitors, in which DHFRand/or TS is the cytotoxic target. Preclinical and clinical trials ofsuch combinations and other similar combinations have providedsynergistic effects that are superior to either drug alone.

Preclinical studies also have shown that inhibition of multiple RTKs hasshown an increase in survival of mice (Shaheen, R. M. et al., CancerRes., 61:1464-1468, 2001). Thus, it is believed that the use of RTKinhibitors along with cytotoxic or conventional cancer chemotherapeuticagents and/or radiation enhances the efficacy of overall antitumortherapy and prevents regrowth following cessation of therapy (Dancey, J.et al., Nat. Rev. Drug Discov., 2:296-313, 2003; Kerbel, R. et al., Nat.Rev. Cancer, 2:727-739, 2002).

RTKs generally are transmembrane receptors consisting of anextracellular growth factor binding domain, a hydrophobic transmembranedomain, and a cytoplasmic domain. The cytoplasmic domain containsregulatory regions and a catalytic tyrosine kinase domain with a bindingsite for both ATP and substrates allowing for autophosphorylation, whichis critical for signal transduction and angiogenesis.

Recently, VEGFR-2 and PDGFR-0, two RTKs, have been implicated incontrolling angiogenesis at two different stages of the angiogenicprocess. In addition, inhibition of VEGFR-2 and PDGFR-0 with twoseparate inhibitors, SU5416 and SU6668, respectively, has been shown toproduce a synergistic effect in early stage as well as late stagepancreatic islet cancer in mouse models by attacking the angiogenicprocess at two different sites (Bergers, G. et al., J. Clin. Invest.,111:1287-1295, 2003; Erber, R. et al., FASEB J., 18:338-340, 2004).

DHFR carries out the reduction of dihydrofolate to tetrahydrofolate(THF), which is utilized by serinehydroxymethyltransferase to produce5,10-methylene-tetrahydrofolate (5,10-CH₂THF). The cofactor 5,10-CH₂THFserves as the source of the methyl group in the conversion ofdeoxyuridine monophosphate to thymidylate catalyzed by thymidylatesynthase (TS). Both TS and DHFR inhibitors are well-establishedcytotoxic agents used in cancer chemotherapy (Gangjee, A. et al., Curr.Pharm. Des., 2:263-280, 1996). Methotrexate and trimetrexate areexamples of such classical and non-classical antifolates, respectively,and 5-fluorouracil and pemetrexed are examples of TS inhibitors usedclinically.

U.S. Pat. No. 5,679,683 discloses 4-substituted aminobenzothieno[3,2-d]pyrimidine and 4-substituted pyrrolo[2,3-d]pyrimidineinhibitors of epidermal growth factor receptor family of tyrosines.

Showalter, H. D. H. et al. (J. Med. Chem., 42:5464-5474, 1999) disclose6,5,6-tricyclic benzothienol[3,2-d]pyrimidines and pyrimido[5,4-b]- and-[4,5-b]indoles as inhibitors of epidermal growth factor receptortyrosine kinases.

PCT published patent application No. WO2005/042500 disclosesarylindenopyridines and arylindenopyridines and their use as anadenosine a2a receptor antagonist.

Gangjee, A. et al. (Bioorganic and Medicinal Chem., 13:5475-5491. 2005)disclose 5-substituted, 2,4-diaminofuro[2,3-d]pyrimidines asmultireceptor tyrosine kinase and dihydrofolate reductase inhibitorswith antiangiogenic and antitumor activity.

In general, it is highly desirable to develop new anti-angiogenic andanti-tumor compounds which inhibit both the formation of new bloodvessels as well as selectively kill tumor cells. There is a need,therefore, for single compounds which provide the desired enzymeinhibition to achieve both cytostatic and cytotoxic activity with a highdegree of selectivity and low toxicity.

SUMMARY OF THE INVENTION

The present invention meets the above need by providing single tricycliccompounds having cytostatic and cytotoxic activity in a single moleculeso that significant drawbacks of different aspects of drug transport oftwo or more drugs to their targets, additive or synergistic toxicitiesof two or more different drugs, resistance of cancer cells to aparticular drug, as well as the cost associated with two or more drugs,is circumvented.

It is an object of the present invention to provide single compoundsthat exhibit anti-angiogenic and anti-tumor activity in tumor cells,such as, without limitation, leukemia, non-small cell lung cancer, coloncancer, central nervous system cancer, melanoma, ovarian cancer, renalcancer, prostate cancer and breast cancer; and other proliferativediseases and disorders such as, without limitation, macular degenerationand retinopathies.

It is a further object of the present invention to provide singlecompounds that possess both cytostatic and cytotoxic activity in tumorcells and other proliferative diseases and disorders.

It is another object of the present invention to provide singlecompounds that possess both cytostatic and cytotoxic activity in tumorcells and other proliferative diseases and disorders by inhibitingactivity against receptor tyrosine kinases (RTKs), dihydrofolatereductase (DHFR), thymidylate synthase (TS) and/or dihydroorotatedehydrogenase (DHODH).

It is another object of the present invention to provide singlecompounds that possess both cytostatic and cytotoxic activity in tumorcells and other proliferative diseases and disorders to overcomepharmacokinetic and pharmacodynamic drawbacks of drug transport of twoor more separate agents to their targets.

It is another object of the present invention to provide singlecompounds that possess both cytostatic and cytotoxic activity in tumorcells and other proliferative diseases and disorders to overcomeadditive or synergistic combined toxicities of using two or moreseparate agents.

It is another object of the present invention to provide singlecompounds that possess both cytostatic and cytotoxic activity in tumorcells and other proliferative diseases and disorders to lessen theresistance of cancer cells to a particular drug.

It is another object of the present invention to provide singlecompounds that possess both cytostatic and cytotoxic activity in tumorcells and other proliferative diseases and disorders to overcome thecost associated with the use of two or more drugs.

It is another object of the present invention to provide methods ofadministering single compounds that possess both cytostatic andcytotoxic activity in tumor cells and other proliferative diseases anddisorders.

The present invention fulfills the above objectives by providing singlecompounds having a combinatorial chemotherapeutic potential of bothcytostatic and cytotoxic activity.

In an aspect of the present invention, there is provided a compound offormula I:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4b-8a, 5-6 and 7-8; the C ring may havean N or substituted N depending on the saturation level of the C ring,and the substitution may be all of R₁, R₂ and R₃;

X and/or Y=N, NH, O, S, C; P═NR₄, O, S, CR₄R₅, wherein R₄ and R₅=loweralkyl, alkene, alkyne, and all of R₁ and R₂;

R₁ and/or R₂=H, alkyl, a cycloalkyl having 6 or less carbons, alkene,alkyne, carbonyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl such as benzene, pyridine, biphenyl, bipyridine, quinazoline,isoquinoline, alkylaryl, alkylheteroaryl, substituted alkylaryl,alkylheteroaryl or a substituted or unsubstituted saturated heterocyclichaving 6 or less atoms;

Z=S, O, NR₅, CR₆R₇, S—C, C—S, O—CR₆, CR₆—O, NR₆—C, C—NR₆, CR₆—NR₇ orCR₆R₇, wherein R₅, R₆, R₇═H or a lower alkyl, alkene, alkyne orcycloalkyl having 6 or less C atoms;

wherein Z may be attached to the C ring at positions 5, 6, 7 or 8 andmay be the same or different and be attached to one or more positions onthe ring;

wherein Z may be zero and R₃ may be directly attached to the C-ring atpositions 5, 6, 7, and/or 8;

wherein when the C-ring is saturated or partially saturated thesubstituted Z or R₃ creates chirality when P═C and R₆ and R₇ aredifferent, then all stereoisomers thereof both separately and as racemicand/or diastereoisomeric mixtures are included;

R₃=H, alkyl, cycloalkyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl, alkylaryl, alkylheteroaryl and substituted saturated orunsaturated alkylheteroaryl and alkylheterocyclic, alkylaryl, p-, m-,o-benzoyl-L-glutamate or 2,5-, 2,4-thienoyl-L-glutamate when the benzeneand thiophene ring may or may not have additional substitutions such asF, mono-, bi- and tricyclic aryl, heteroaryl or combinations thereof,ring substitutions such as biphenyl, bipyridyl or a phenyl-pyridyl orfused such as a quinoline or naphthyl including substituted systems suchas a 2-chloro,4-biphenyl and tricyclic and substituted tricyclicsystems.

In another aspect of the present invention, there is provided a compoundof formula II:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4b-8a, 5-6 and 7-8; the C ring may havean N or substituted N depending on the saturation level of the C ring,and the substitution may be all of R₁, R₂ and R₃;

X and/or Y=N, NH, O, S, C; P═NR₄, O, S, CR₄R₅, wherein R₄ and R₅=loweralkyl, alkene, alkyne, and all of R₁ and R₂;

R₁ and/or R₂=H, alkyl, a cycloalkyl having 6 or less carbons, alkene,alkyne, carbonyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl such as benzene, pyridine, biphenyl, bipyridine, quinazoline,isoquinoline, alkylaryl, alkylheteroaryl, substituted alkylaryl,alkylheteroaryl or a substituted or unsubstituted saturated heterocyclichaving 6 or less atoms;

Z=S, O, NR₅, CR₆R₇, S—C, C—S, O—CR₆, CR₆—O, NR₆—C, C—NR₆, CR₆—NR₇ orCR₆R₇, wherein R₅, R₆, R₇═H or a lower alkyl, alkene, alkyne orcycloalkyl having 6 or less C atoms;

wherein Z may be attached to the C ring at positions 5, 6, 7 or 8 andmay be the same or different and be attached to one or more positions onthe ring;

wherein Z may be zero and R₃ may be directly attached to the C-ring atpositions 5, 6, 7, and/or 8;

wherein when the C-ring is saturated or partially saturated thesubstituted Z or R₃ creates chirality when P═C and R₆ and R₇ aredifferent, then all stereoisomers thereof both separately and as racemicand/or diastereoisomeric mixtures are included;

R₃=H, alkyl, cycloalkyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl, alkylaryl, alkylheteroaryl and substituted saturated orunsaturated alkylheteroaryl and alkylheterocyclic, alkylaryl, p-, m-,o-benzoyl-L-glutamate or 2,5-, 2,4-thienoyl-L-glutamate when the benzeneand thiophene ring may or may not have additional substitutions such asF, mono-, bi- and tricyclic aryl, heteroaryl or combinations thereof,ring substitutions such as biphenyl, bipyridyl or a phenyl-pyridyl orfused such as a quinoline or naphthyl including substituted systems suchas a 2-chloro, 4-biphenyl and tricyclic and substituted tricyclicsystems.

In another aspect of the present invention, there is provided a methodof inhibiting receptor tyrosine kinase(s), dihydrofolate reductase,thymidylate synthase and/or dihydroorotate dehydrogenase activity in ananimal or human in need thereof, comprising administering to said animalor human a therapeutically effective amount in unit dosage form of acompound of formula I:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4b-8a, 5-6 and 7-8; the C ring may havean N or substituted N depending on the saturation level of the C ring,and the substitution may be all of R₁, R₂ and R₃;

X and/or Y=N, NH, O, S, C; P═NR₄, O, S, CR₄R₅, wherein R₄ and R₅=loweralkyl, alkene, alkyne, and all of R₁ and R₂;

R₁ and/or R₂=H, alkyl, a cycloalkyl having 6 or less carbons, alkene,alkyne, carbonyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl such as benzene, pyridine, biphenyl, bipyridine, quinazoline,isoquinoline, alkylaryl, alkylheteroaryl, substituted alkylaryl,alkylheteroaryl or a substituted or unsubstituted saturated heterocyclichaving 6 or less atoms;

Z=S, O, NR₅, CR₆R₇, S—C, C—S, O—CR₆, CRr—O, NR₆—C, C—NR₆, CR₆—NR₇ orCR₆R₇, wherein R₅, R₆, R₇═H or a lower alkyl, alkene, alkyne orcycloalkyl having 6 or less C atoms;

wherein Z may be attached to the C ring at positions 5, 6, 7 or 8 andmay be the same or different and be attached to one or more positions onthe ring;

wherein Z may be zero and R₃ may be directly attached to the C-ring atpositions 5, 6, 7, and/or 8;

wherein when the C-ring is saturated or partially saturated thesubstituted Z or R₃ creates chirality when P═C and R₆ and R₇ aredifferent, then all stereoisomers thereof both separately and as racemicand/or diastereoisomeric mixtures are included;

R₃=H, alkyl, cycloalkyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl, alkylaryl, alkylheteroaryl and substituted saturated orunsaturated alkylheteroaryl and alkylheterocyclic, alkylaryl, p-, m-,o-benzoyl-L-glutamate or 2,5-, 2,4-thienoyl-L-glutamate when the benzeneand thiophene ring may or may not have additional substitutions such asF, mono-, bi- and tricyclic aryl, heteroaryl or combinations thereof,ring substitutions such as biphenyl, bipyridyl or a phenyl-pyridyl orfused such as a quinoline or naphthyl including substituted systems suchas a 2-chloro,4-biphenyl and tricyclic and substituted tricyclicsystems.

In another aspect of the present invention, there is provided a methodof inhibiting receptor tyrosine kinase(s), dihydrofolate reductase,thymidylate synthase and/or dihydroorotate dehydrogenase activity in ananimal or human in need thereof, comprising administering to said animalor human a therapeutically effective amount in unit dosage form of acompound of formula II:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4b-8a, 5-6 and 7-8; the C ring may havean N or substituted N depending on the saturation level of the C ring,and the substitution may be all of R₁, R₂ and R₃;

X and/or Y=N, NH, O, S, C; P═NR₄, O, S, CR₄R₅, wherein R₄ and R₅=loweralkyl, alkene, alkyne, and all of R₁ and R₂;

R₁ and/or R₂=H, alkyl, a cycloalkyl having 6 or less carbons, alkene,alkyne, carbonyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl such as benzene, pyridine, biphenyl, bipyridine, quinazoline,isoquinoline, alkylaryl, alkylheteroaryl, substituted alkylaryl,alkylheteroaryl or a substituted or unsubstituted saturated heterocyclichaving 6 or less atoms;

Z=S, O, NR₅, CR₆R₇, S—C, C—S, O—CR₆, CR₆—O, NR₆—C, C—NR₆, CR₆—NR₇ orCR₆R₇, wherein R₅, R₆, R₇═H or a lower alkyl, alkene, alkyne orcycloalkyl having 6 or less C atoms;

wherein Z may be attached to the C ring at positions 5, 6, 7 or 8 andmay be the same or different and be attached to one or more positions onthe ring;

wherein Z may be zero and R₃ may be directly attached to the C-ring atpositions 5, 6, 7, and/or 8;

wherein when the C-ring is saturated or partially saturated thesubstituted Z or R₃ creates chirality when P═C and R₆ and R₇ aredifferent, then all stereoisomers thereof both separately and as racemicand/or diastereoisomeric mixtures are included;

R₃=H, alkyl, cycloalkyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl, alkylaryl, alkylheteroaryl and substituted saturated orunsaturated alkylheteroaryl and alkylheterocyclic, alkylaryl, p-, m-,o-benzoyl-L-glutamate or 2,5-, 2,4-thienoyl-L-glutamate when the benzeneand thiophene ring may or may not have additional substitutions such asF, mono-, bi- and tricyclic aryl, heteroaryl or combinations thereof,ring substitutions such as biphenyl, bipyridyl or a phenyl-pyridyl orfused such as a quinoline or naphthyl including substituted systems suchas a 2-chloro,4-biphenyl and tricyclic and substituted tricyclicsystems.

BRIEF DESCRIPTION OF THE DRAWINGS

A full understanding of the invention can be gained from the followingdescription of the preferred embodiments when read in conjunction withthe accompanying drawings in which:

FIG. 1 illustrates primary tumor growth of COLO-205 metastatic humancolon cancer cells implanted into the flank of athymic mice in responseto DMBI (PDGFR kinase inhibitor) and AAG143-43 given at 25 mg/kg 3×weekly (M, W, F).

FIG. 2 illustrates vascularity of COLO-205 primary tumors in athymicmice in response to DMBI (PDGFR kinase inhibitor) and AAG148-43 given at25 mg/kg 3× weekly (M, W, F).

FIG. 3 illustrates metastasis to the liver of COLO-205 cells implantedinto athymic mice in response to DMBI (PDGFR kinase inhibitor) andAAG148-43 given at 25 mg/kg 3× weekly (M, W, F).

FIG. 4 is a dose-response curve of percentage growth for each of thecell lines shown in Table 4 of the specification.

FIG. 5 are dose response curves for each of the cancer types shown inTable 4 of the specification.

FIG. 6 shows mean graphs for each of the cancer types and correspondingcell lines shown in Table 4 of the specification.

FIG. 7A, B illustrate tumor parameters of primary B16-F10 tumors inathymic mice in response to drugs given at 25 mg/kg 3× weekly (M, W, F).FIG. 7A is a graph of tumor volume. FIG. 7B is a bar graph of tumorgrowth rate.

FIG. 8 illustrates metastasis of B16-F10 tumors to the lung in responseto drugs given at 25 mg/kg 3× weekly (M, W, F).

FIG. 9 illustrates vascularity of B16-F10 tumors in athymic mice inresponse to drugs given at 25 mg/kg 3× weekly (M, W, F).

FIG. 10 shows elemental analysis for Compounds 4, 12-15 and 19.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides tricyclic compounds having cytostatic andcytotoxic activity in a single molecule having receptor tyrosinekinase(s) (RTK), dihydrofolate reductase (DHFR), thymidylate synthase(TS) and/or dihydroorotate dehydrogenase (DHODH) inhibitory activity andmethods of use thereof.

In an embodiment of the present invention, there is provided a compoundof formula I:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4b-8a, 5-6 and 7-8; the C ring may havean N or substituted N depending on the saturation level of the C ring,and the substitution may be all of R₁, R₂ and R₃;

X and/or Y=N, NH, O, S, C; P═NR₄, O, S, CR₄R₅, wherein R₄ and R₅=loweralkyl, alkene, alkyne, and all of R₁ and R₂;

R₁ and/or R₂=H, alkyl, a cycloalkyl having 6 or less carbons, alkene,alkyne, carbonyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl such as benzene, pyridine, biphenyl, bipyridine, quinazoline,isoquinoline, alkylaryl, alkylheteroaryl, substituted alkylaryl,alkylheteroaryl or a substituted or unsubstituted saturated heterocyclichaving 6 or less atoms;

Z=S, O, NR₅, CR₆R₇, S—C, C—S, O—CR₆, CR₆—O, NR₆—C, C—NR₆, CR₆—NR₇ orCR₆R₇, wherein R₅, R₆, R₇=H or a lower alkyl, alkene, alkyne orcycloalkyl having 6 or less C atoms;

wherein Z may be attached to the C ring at positions 5, 6, 7 or 8 andmay be the same or different and be attached to one or more positions onthe ring;

wherein Z may be zero and R₃ may be directly attached to the C-ring atpositions 5, 6, 7, and/or 8;

wherein when the C-ring is saturated or partially saturated thesubstituted Z or R₃ creates chirality when P═C and R₆ and R₇ aredifferent, then all stereoisomers thereof both separately and as racemicand/or diastereoisomeric mixtures are included;

R₃=H, alkyl, cycloalkyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl, alkylaryl, alkylheteroaryl and substituted saturated orunsaturated alkylheteroaryl and alkylheterocyclic, alkylaryl, p-, m-,o-benzoyl-L-glutamate or 2,5-, 2,4-thienoyl-L-glutamate when the benzeneand thiophene ring may or may not have additional substitutions such asF, mono-, bi- and tricyclic aryl, heteroaryl or combinations thereof,ring substitutions such as biphenyl, bipyridyl or a phenyl-pyridyl orfused such as a quinoline or naphthyl including substituted systems suchas a 2-chloro,4-biphenyl and tricyclic and substituted tricyclicsystems.

A preferred form of formula I is where Y=NH; R₂=H; P═NR₄; R₄=H; X=NH;R₁=H; Z=S and R₃=a phenyl:

Another preferred form of formula I is where Y=NH; R₂=H; P═CR₄R₅; R₄=H;R₅=H; X=NH; R₁=p-chlorophenyl; Z=0; and R₃=H. The substituted aryl canbe a halide, such as a chloride atom. A suitable ring structure is:

In another aspect of the present invention, there is provided a compoundof formula II:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4b-8a, 5-6 and 7-8; the C ring may havean N or substituted N depending on the saturation level of the C ring,and the substitution may be all of R₁, R₂ and R₃;

X and/or Y=N, NH, O, S, C; P═NR₄, O, S, CR₄R₅, wherein R₄ and R₅=loweralkyl, alkene, alkyne, and all of R₁ and R₂;

R₁ and/or R₂=H, alkyl, a cycloalkyl having 6 or less carbons, alkene,alkyne, carbonyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl such as benzene, pyridine, biphenyl, bipyridine, quinazoline,isoquinoline, alkylaryl, alkylheteroaryl, substituted alkylaryl,alkylheteroaryl or a substituted or unsubstituted saturated heterocyclichaving 6 or less atoms;

X=S, O, NR₅, CR₆R₇, S—C, C—S, O—CR₆, CR₆—O, NR₆—C, C—NR₆, CR₆—NR₇ orCR₆R₇, wherein R₅, R₆, R₇=H or a lower alkyl, alkene, alkyne orcycloalkyl having 6 or less C atoms;

wherein Z may be attached to the C ring at positions 5, 6, 7 or 8 andmay be the same or different and be attached to one or more positions onthe ring;

wherein Z may be zero and R₃ may be directly attached to the C-ring atpositions 5, 6, 7, and/or 8;

wherein when the C-ring is saturated or partially saturated thesubstituted Z or R₃ creates chirality when P═C and R₆ and R₇ aredifferent, then all stereoisomers thereof both separately and as racemicand/or diastereoisomeric mixtures are included;

R₃=H, alkyl, cycloalkyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl, alkylaryl, alkylheteroaryl and substituted saturated orunsaturated alkylheteroaryl and alkylheterocyclic, alkylaryl, p-, m-,o-benzoyl-L-glutamate or 2,5-, 2,4-thienoyl-L-glutamate when the benzeneand thiophene ring may or may not have additional substitutions such asF, mono-, bi- and tricyclic aryl, heteroaryl or combinations thereof,ring substitutions such as biphenyl, bipyridyl or a phenyl-pyridyl orfused such as a quinoline or naphthyl including substituted systems suchas a 2-chloro,4-biphenyl and tricyclic and substituted tricyclicsystems.

As used herein, the term “lower alkyl” group refers to those lower alkylgroups having one to about six carbon atoms, such as for example methyl,ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl,cyclohexyl, cyclopropylmethyl or cyclobutylmethyl groups. Alkyl groupssharing one to about six carbon atoms are preferred. These lower alkylgroups are straight chain, branched chain or cyclic (alicyclichydrocarbon) arrangements. The carbon atoms of these straight chain,branched chain or cyclic arranged alkyl groups may have one or moresubstituents for the hydrogens attached to the carbon atoms.

As used herein, the terms “heteroalkyl” and “heteroalkenyl” will be usedto refer to alkyl or alkene chains from one to about 3 atoms where oneor more of the carbons has been replaced with nitrogen, oxygen orsulfur. Thus “heteroalkyl” and “heteroalkenyl” groups will include, forexample, C—C—N, C—S, S—C, C—O, C—C—O, O—C, N—C—C, N—C═C and othervarious combinations, as will be apparent to one skilled in the art. Theabove list is not meant to be exhaustive, and many combinations arecontemplated as within the scope of the present invention.

“Aryl” groups, as used herein, will refer to compounds whose moleculeshave an aromatic ring structure, such as the six-carbon ring of benzene,or multiple rings which are either fused or unfused, such as condensedsix-carbon rings of other aromatic derivatives. The term “aryl” is alsodefined to include diaryl, triaryl and polyaryl groups, which would havetwo, three or more rings, respectively. Thus, suitable aryl groups wouldinclude, for example, phenyl, biphenyl, naphthyl, phenanthrene,anthracene groups and aryl oxyaryl groups. This list is not meant to beexhaustive, and any aryl group, as these terms are defined above andcommonly understood in the art, are within the scope of the presentinvention.

The term “heteroaryl”, as used herein, will be used to refer to aromaticring structures having at least one atom in the ring which is notcarbon, such as oxygen, nitrogen or sulfur. “Heteroaryls” as used hereinalso refers to aromatic ring structures that are part of larger ringstructures, such as two or three member ring systems, which may be fusedor unfused, in which one of the rings is as described above. Thus,“heteroaryl” can refer to ring systems in which one or more ringscontain a heteroatom and one or more rings do not. It will be understoodthat this list is not meant to be exhaustive, and that any heteroarylgroup, as these terms are defined above and commonly understood in theart, are within the scope of the present invention. Examples include butare not limited to pyrroles, thiophenes, furans, imidazoles, and thelike, as well as fused ring structures having rings of different sizes,such as benzofurans, indoles, purines, and the like.

Also included within the scope of the present invention are alicyclicgroups, as that term is understood in the art, and heterocyclic groups.As used herein, the term “heterocyclic group” will refer to non-aromaticcyclic substituents in which one or more members of the ring is notcarbon, for example oxygen, sulfur or nitrogen.

The terms “alkylaryl” (or “alkaryl”) or “alkylheteroaryl” as used hereinwill refer to groups having an alkyl moiety attached to an aryl orheteroaryl ring. The alkyl moiety is preferably a straight, branched orcyclic alkyl group having one to about six carbon atoms. This alkylmoiety may also contain oxygen, nitrogen or sulfur atoms, and cantherefore be an alkoxy group. The aryl or heteroaryl moiety of thealkylaryl group is a substituted or unsubstituted aryl or heteroarylgroup, as these terms are described above. As used herein, the terms“alkylaryl” or “alkylheteroaryl” will also be used to refer to arylalkylgroups or heteroarylalkyl groups, as those terms are understood in theart, and will denote attachment of such a substituent at either thealkyl or the aryl portion of the group. Thus, for example, a benzylgroup would be embraced by the term “alkylaryl”.

Any of the cyclic substituents described above, such as the aryl,heteroaryl, alkylaryl, alkylheteroaryl, alicyclic, or heterocyclicgroups are optionally substituted with one or more substituents aslisted above. In the case of more than one substituent, the substituentsare independently selected. “Alkoxy groups” and “alkyl groups” includestraight or branched chains having up to about six members. “Halogen”refers to chlorine, bromine, iodine and fluorine. “Aryl and heteroarylgroups” are as described above. When a carboxylic acid is a substituent,it will be appreciated that the moiety represents an acid such asbenzoic acid.

“Acyl” refers to an organic acid group in which the OH is replaced bysome other substituent, and is generally designated as RCO— where R is aC₁₋₆ alkyl, C₂₋₆ alkenyl, or C₂₋₆ alkynyl straight or branched chaingroup.

As used herein, the terms “aroyl” or “heteroaroyl”, such as when usedwithin the term p-aroyl-L-glutamate, refers to benzoyl, napthoyl,thiophenoyl, furophenoyl, pyrroyl, and any other “aroyl” or“heteroaroyl” as these terms would be understood by one skilled in theart. “Aroyl” and “heteroaroyl” are generally defined in the art as anaromatic or heteroaromatic compound having a carbonyl moiety.“Glutamate” will be understood as representing both the ester form(glutamate) and the acid form (glutamic acid).

It will appreciated by those skilled in the art that a general formuladepicting compounds having side chains with adjacent carbons having adouble bond will result in both cis and trans isomers as possiblestructures. Both the cis and trans isomers, and mixtures thereof, of anysuch compound within the broad general formula described in formulas Iand II are contemplated as being within the present invention.

A preferred form of formula II is where Y=NH; R₂=H; P═CR₄R₅; R₄=H; R₅=H;X=NH; R₁=phenyl; Z=0; and R₃=H:

In another embodiment of the present invention, there is provided amethod of inhibiting receptor tyrosine kinase(s), dihydrofolatereductase, thymidylate synthase and/or dihydroorotate dehydrogenaseactivity in an animal or human in need thereof, comprising administeringto said animal or human a therapeutically effective amount in unitdosage form of a compound of formula I:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4b-8a, 5-6 and 7-8; the C ring may havean N or substituted N depending on the saturation level of the C ring,and the substitution may be all of R₁, R₂ and R₃;

X and/or Y=N, NH, O, S, C; P═NR₄, O, S, CR₄R₅, wherein R₄ and R₅=loweralkyl, alkene, alkyne, and all of R₁ and R₂;

R₁ and/or R₂=H, alkyl, a cycloalkyl having 6 or less carbons, alkene,alkyne, carbonyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl such as benzene, pyridine, biphenyl, bipyridine, quinazoline,isoquinoline, alkylaryl, alkylheteroaryl, substituted alkylaryl,alkylheteroaryl or a substituted or unsubstituted saturated heterocyclichaving 6 or less atoms;

Z=S, O, NR₅, CR₆R₇, S—C, C—S, O—CR₆, CR₆—O, NR₆—C, C—NR₆, CR₆—NR₇ orCR₆R₇,

wherein R₅, R₆, R₇=H or a lower alkyl, alkene, alkyne or cycloalkylhaving 6 or less C atoms;

wherein Z may be attached to the C ring at positions 5, 6, 7 or 8 andmay be the same or different and be attached to one or more positions onthe ring;

wherein Z may be zero and R₃ may be directly attached to the C-ring atpositions 5, 6, 7, and/or 8;

wherein when the C-ring is saturated or partially saturated thesubstituted Z or R₃ creates chirality when P═C and R₆ and R₇ aredifferent, then all stereoisomers thereof both separately and as racemicand/or diastereoisomeric mixtures are included;

R₃=H, alkyl, cycloalkyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl, alkylaryl, alkylheteroaryl and substituted saturated orunsaturated alkylheteroaryl and alkylheterocyclic, alkylaryl, p-, m-,o-benzoyl-L-glutamate or 2,5-, 2,4-thienoyl-L-glutamate when the benzeneand thiophene ring may or may not have additional substitutions such asF, mono-, bi- and tricyclic aryl, heteroaryl or combinations thereof,ring substitutions such as biphenyl, bipyridyl or a phenyl-pyridyl orfused such as a quinoline or naphthyl including substituted systems suchas a 2-chloro,4-biphenyl and tricyclic and substituted tricyclicsystems.

Proliferative diseases and/or disorders that may be treated according tothe methods of the present invention include, without limitation,leukemia, non-small cell lung cancer, colon cancer, central nervoussystem cancer, melanoma, ovarian cancer, renal cancer, prostate cancer,breast cancer; macular degeneration and retinopathies.

A preferred form of formula I is where Y=NH; R₂=H; P═NR₄; R₄=H; X=NH;R₁=H; Z=S and R₃=a phenyl:

Another preferred form of formula I is where Y=NH; R₂=H; P═CR₄R₅; R₄=H;R₅=H; X=NH; R₁=p-chlorophenyl; Z=0; and R₃=H. The substituted aryl canbe a halide, such as a chloride atom. A suitable ring structure is:

In another embodiment of the present invention, there is provided amethod of inhibiting receptor tyrosine kinase(s), dihydrofolatereductase, thymidylate synthase and/or dihydroorotate dehydrogenaseactivity in an animal or human in need thereof, comprising administeringto said animal or human a therapeutically effective amount in unitdosage form of a compound of formula II:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4b-8a, 5-6 and 7-8; the C ring may havean N or substituted N depending on the saturation level of the C ring,and the substitution may be all of R₁, R₂ and R₃;

X and/or Y=N, NH, O, S, C; P═NR₄, O, S, CR₄R₅, wherein R₄ and R₅=loweralkyl, alkene, alkyne, and all of R₁ and R₂;

R₁ and/or R₂=H, alkyl, a cycloalkyl having 6 or less carbons, alkene,alkyne, carbonyl, carbonyl, aryl, heteroaryl, substituted aryl,substituted heteroaryl such as benzene, pyridine, biphenyl, bipyridine,quinazoline, isoquinoline, alkylaryl, alkylheteroaryl, substitutedalkylaryl, alkylheteroaryl or a substituted or unsubstituted saturatedheterocyclic having 6 or less atoms;

Z=S, O, NR₅, CR₆R₇, S—C, C—S, O—CR₆, CR₆—O, NR₆—C, C—NR₆, CR₆—NR₇ orCR₆R₇, wherein R₅, R₆, R₇═H or a lower alkyl, alkene, alkyne orcycloalkyl having 6 or less C atoms;

wherein Z may be attached to the C ring at positions 5, 6, 7 or 8 andmay be the same or different and be attached to one or more positions onthe ring;

wherein Z may be zero and R₃ may be directly attached to the C-ring atpositions 5, 6, 7, and/or 8;

wherein when the C-ring is saturated or partially saturated thesubstituted Z or R₃ creates chirality when P═C and R₆ and R₇ aredifferent, then all stereoisomers thereof both separately and as racemicand/or diastereoisomeric mixtures are included;

R₃═H, alkyl, cycloalkyl, aryl, heteroaryl, substituted aryl, substitutedheteroaryl, alkylaryl, alkylheteroaryl and substituted saturated orunsaturated alkylheteroaryl and alkylheterocyclic, alkylaryl, p-, m-,o-benzoyl-L-glutamate or 2,5-, 2,4-thienoyl-L-glutamate when the benzeneand thiophene ring may or may not have additional substitutions such asF, mono-, bi- and tricyclic aryl, heteroaryl or combinations thereof,ring substitutions such as biphenyl, bipyridyl or a phenyl-pyridyl etc.or fused such as a quinoline or naphthyl including substituted systemssuch as a 2-chloro,4-biphenyl and tricyclic and substituted tricyclicsystems.

A preferred form of formula II is where Y=NH; R₂=H; P═CR₄R₅; R₄=H; R₅=H;X=NH; R₁=phenyl; Z=0; and R₃=H:

As used herein, the term “patient” means adult members of the animalkingdom, including, but not limited to, human beings.

As used herein, the term “therapeutically effective amount” refers tothat amount of any of the present compounds required to bring about adesired effect in a patient. The desired effect will vary depending onthe illness being treated. For example, the desired effect may bereducing tumor size, destroying cancerous cells, preventing metastasisor reducing symptoms associated with the various other diseases listedabove and contemplated as being within the treatment methods of thepresent invention. On its most basic level, a therapeutically effectiveamount is that amount needed to inhibit the activity of receptortyrosine kinase(s) generally and/or dihydrofolate reductase and/orthymidylate synthase and/or dihydroorotate dehydrogenase. Any amount ofinhibition will yield a benefit to a patient and is therefore within thescope of the invention.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the patients being treated, each unitcontaining a predetermined quantity or effective amount of a tricycliccompound to produce the desired effect in association with apharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the particularcompound and the particular effect to be achieved.

Compounds containing formula I or formula II can be administered to ananimal or human via various routes including parenterally, orally orintraperitoneally. Parenteral administration includes the followingroutes: intravenous; intramuscular; interstitial, intraarterial;subcutaneous; intraocular; intracranial; intraventricular;intrasynovial; transepithelial, including transdermal, pulmonary viainhalation, ophthalmic, sublingual and buccal; topical, includingdermal, ocular, rectal, or nasal inhalation via insufflation ornebulization.

Compounds containing formula I or formula II that are orallyadministered can be enclosed in hard or soft shell gelatin capsules, orcompressed into tablets. Compounds also can be incorporated with anexcipient and used in the form of ingestible tablets, buccal tablets,troches, capsules, sachets, lozenges, elixirs, suspensions, syrups,wafers and the like. Compounds containing formula I or formula II can bein the form of a powder or granule, a solution or suspension in anaqueous liquid or non-aqueous liquid, or in an oil-in-water emulsion.

The tablets, troches, pills, capsules and the like also can contain, forexample, a binder, such as gum tragacanth, acacia, corn starch; gelatingexcipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; a sweetening agent, such as sucrose, lactose orsaccharin; or a flavoring agent. When the dosage unit form is a capsule,it can contain, in addition to the materials described above, a liquidcarrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For example,tablets, pills, or capsules can be coated with shellac, sugar or both. Asyrup or elixir can contain the active compound, sucrose as a sweeteningagent, methyl and propylparabens as preservatives, a dye and flavoring.Any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic. Additionally, thecompounds of formula I or formula II can be incorporated intosustained-release preparations and formulations.

The compounds of formula I or formula II can be administered to thecentral nervous system, parenterally or intraperitoneally. Solutions ofthe compound as a free base or a pharmaceutically acceptable salt can beprepared in water mixed with a suitable surfactant, such ashydroxypropylcellulose. Dispersions also can be prepared in glycerol,liquid polyethylene glycols and mixtures thereof, and in oils. Underordinary conditions of storage and use, these preparations can contain apreservative and/or antioxidants to prevent the growth of microorganismsor chemical degeneration.

The pharmaceutical forms suitable for injectable use include, withoutlimitation, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It can be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium which contains, forexample, water, ethanol, polyol (such as propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, or vegetable oils. Theproper fluidity can be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size (inthe case of a dispersion) and by the use of surfactants. The preventionof the action of microorganisms can be brought about by variousantibacterial and anti-fungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride.

Sterile injectable solutions are prepared by incorporating the compoundof formula I or formula II in the required amount in the appropriatesolvent with various of the other ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the sterilized compound of formula I orformula II into a sterile vehicle that contains the basic dispersionmedium and any of the other ingredients from those enumerated above. Inthe case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze drying.

Pharmaceutical compositions which are suitable for administration to thenose and buccal cavity include, without limitation, self-propelling andspray formulations, such as aerosol, atomizers and nebulizers.

The therapeutic compounds of formula I and formula II can beadministered to an animal or human alone or in combination withpharmaceutically acceptable carriers or as pharmaceutically acceptablesalts, the proportion of which is determined by the solubility andchemical nature of the compound, chosen route of administration andstandard pharmaceutical practice.

The compounds of the present invention are distinguished from prior artcompounds in that the inventive compounds disclosed herein havesubstituents on the C-ring which are either halogens such as chloro,bromo, iodo and fluoro; L-glutamate-bearing side chains; aryl (directlyconnected); or one atom linked aryl of the type: thioaryl; aminoaryl;oxoaryl; carboaryl; compounds in which the C-ring is partiallysubstituted; compounds of the type in which the B-ring is cyclopentane;compounds in which the substituents on the B-ring are either aryl ormethyl aryl (e.g., benzyl).

The present invention is more particularly described in the followingnon-limiting examples, which are intended to be illustrative only, asnumerous modifications and variations therein will be apparent to thoseskilled in the art.

EXAMPLES Example 1 Compounds Derived from Compound of Formula I 1.AAG148-43 Efficacy in an Animal Model of Cancer

Single compounds which possess both antiangiogenic, i.e., cytostatic,activity and cytotoxic activity were designed, synthesized andevaluated. COLO-205 metastatic human colon cancer cells were implantedin mice. AAG-148-43 was administered to the mice at a dose of 25 mg/kgthree times a week. FIG. 1 shows the antitumor activity of AAG-148-43compared to DMBI (a standard PDGFR-β inhibitor). AAG-148-43significantly reduced the growth of COLO-205 metastatic human coloncancer cells in mice comparable to the reduction observed in thestandard PDGFR-β inhibitor.

As shown in Table 1, the cytotoxic activity of the compounds resulted inthe inhibition of VEGFR-2 and PDGFR-β at levels comparable to standardsSU5416 and DMBI, respectively. For AAG-148-43 and AAG-148-311, the RTKinhibitory activity (PDGFR-β) activity was compared to the standardDMBI.

TABLE 1

Compound # ID Ar 1 AAG148-43 Ph 2 AAG148-311 4-MePh RTK Inhibitoryactivity IC₅₀ values (μM) of kinase inhibition and A431 cytotoxicityassay VEGFR-2 EGFR kinase kinase PDGFR-β A431 Compd # inhibitioninhibition kinase inhibition cytotoxicity AAG- 15.07 22.6 2.8  49.2148-43 PD153035  0.23 SU5416 12.9 DMBI 3.75 Cisplatin 10.6

As shown in Table 2, the cytotoxic activity resulted in the inhibitionof enzyme(s) in the folate metabolic pathway such as thymidylatesynthase (TS). The TS inhibitory activity was compared to the standardPDDF. AAG-148-43 was a preferred embodiment, having shown multi-RTK(VEGF-2 and PDGFR-β) (cytostatic) inhibitory activity as well as TS(cytotoxic) inhibitory activity.

TABLE 2 TS inhibitory activity IC₅₀ values (μM) of Thymidylate Synthaseinhibition Compd # Human E. coli Toxo AAG-148-43 0.54 >27 (36) 0.11AAG-148-311 0.39 >26 (34) 0.18 PDDF 0.06 0.06 0.06 Pemetrexed 29 15 14Raltitrexed .29 2.3 0.48

FIG. 2 shows the reduction of vascularity of primary tumors withAAG-148-43 compared to DMBI and untreated animals. AAG-148-43 reducedvascularity much better than DMBI, and thus demonstrated its potentantiangiogenic activity in tumors in an animal model of cancer.

COLO-205 cells were implanted into athymic mice. FIG. 3 shows metastasisto the liver of the COLO-205 cells in response to administration ofAAG-148-43, DMBI and untreated mice. AAG-148-43 significantly reducedmetastasis to the liver compared to DMBI and the untreated animals,demonstrating the ability of AAG-148-43 to significantly inhibitmetastasis.

The above-described results demonstrate the significantly better overalloutcome for reduction of tumor growth, reduction of vascularity andreduction of metastasis with AAG-148-43 of this series compared to thestandard and the untreated mice. In addition, AAG-148-43 administrationcaused no apparent toxicity (no loss in weight of the animals) at a doseof 25 mg/kg three times weekly.

2. Compound 3 Efficacy in an Animal Model of Cancer

The structure of Compound 3 (AAG145-136; Table 3) is as follows:

-   -   wherein Formula I has the following substitutions: P═CH₂; R₃=H;        X=NH; Y=NH₂; and R₁=p-ClC₆H₄.

Compound 3 was shown to have potent multi-RTK inhibitory activity inEGFR and PDGFR-β along with A431 cytotoxicity and inhibition ofangiogenesis in a CAM assay (Table 3).

TABLE 3 IC₅₀ value (μM) of kinase inhibition, A431 cytotoxicity andinhibition of CAM assay Compound EGFR kinase Flk-1 kinase Flt-1 kinasePDGFR-β kinase A431 cytotoxicity CAM angiogenesis

>200 >200 21.7 126.4 2.12

42.6 11.0 7.8 126.4 14.8

24.1 >200 126.3 >5000 104.3

11.7 197.1 3.6 9.7 3.67

26.0 >200 0.8 9.7 0.82

19.6 133.9 >500 235.0 SU5416 10.6 19.2 ± 4.2 0.032 ± 0.005 PD153035 0.23PD168393 0.13 cisplatin 10.6 DMBI 3.75 VDGF inhibitor 11.9

>200 171.2 43.1 4.9 50.8

>200 43.0 75.5 13.9

>200 55.7 7.1 9.8 78.3

>200 72.9 6.4 158.1 SU5416 10.6 19.2 ± 4.2 0.032 ± 0.005 PD153035 0.23PD168393 0.13 cisplatin 10.6 DMBI 3.75 VEGF inhibitor 11.9

Compound 3 was evaluated in a 60 preclinical in vitro tumor screeningpanel of the National Cancer Institute and showed excellent cytotoxicityin a wide range of tumors with a GI₅₀ from 3.12×10⁻⁸ M to 10⁻⁶ M withGI₅₀ against 10 tumors in the 10⁻⁸ M and GI₅₀ 10⁻⁷ M against 29 tumorcell lines. Compound 3, when compared in a COMPARE ANALYSIS, alsosuggested a mechanism of cytotoxicity via inhibition of dihydrofolatereductase and/or dihydroorotate dehydrogenase (Table 4). FIG. 4 is adose-response curve of percentage growth for each of the cell linesshown in Table 4. FIG. 5 are dose response curves for each of the cancertypes shown in Table 4. FIG. 6 shows mean graphs for each of the cancertypes and corresponding cells lines shown in Table 4.

TABLE 4 National Cancer Institute Developmental Therapeutics ProgramIn-Vitro Testing Results NSC: D-741192/1 Experiment ID: 0702NS13 TestType: 08 Units: Molar Report Date: May 03, 2007 Test Date: Feb. 05, 2007ONS: MC: COMI: ZY/AG 145-136 (45284) Stain Reagent: SRB Dual-PassRelated SSPL: 0D4H Log10 Concentration Time Mean Optical DensitiesPercent Growth Panel/Cell Line Zero Ctrl −8.0 −7.0 −6.0 −5.0 −4.0 −8.0−7.0 −6.0 −5.0 −4.0 GI50 TGI LC50 Leukemia OCRF-CEM 0.390 1.286 1.0830.587 0.462 0.446 0.485 77 22 8 6 11 3.12E−6 >1.00E−4 >1.00E−4 HL-60(TB)0.553 2.179 1.966 1.481 0.921 0.760 0.756 87 56 23 13 121.50E−7 >1.00E−4 >1.00E−4 K-562 0.174 0.986 1.012 0.899 0.333 0.2010.380 103 89 20 16 26 3.56E−7 >1.00E−4 >1.00E−4 MOLT-4 0.473 1.674 1.6640.944 0.637 0.602 0.726 99 39 14 11 21 6.61E−8 >1.00E−4 >1.00E−4RPMI-8226 0.380 0.926 0.903 0.867 0.556 0.441 0.419 96 89 32 11 74.89E−7 >1.00E−4 >1.00E−4 SR 0.421 1.274 1.020 0.768 0.611 0.525 0.44070 41 22 12 2 4.84E−8 >1.00E−4 >1.00E−4 Non-Small Cell Lung CancerAS45/ATCC 0.201 1.071 0.901 0.486 0.382 0.332 0.263 30 33 21 15 74.34E−8 >1.00E−4 >1.00E−4 EKVX 0.181 0.519 0.524 0.478 0.412 0.270 0.194101 68 68 26 4 2.71E−8 >1.00E−4 >1.00E−4 HOP-52 0.518 1.498 1.467 1.4350.911 0.751 0.829 97 94 40 25 32 6.54E−7 >1.00E−4 >1.00E−4 HOP-92 0.7571.144 1.145 1.141 1.051 1.009 0.841 100 96 74 62 151.81E−5 >1.00E−4 >1.00E−4 NCI-H226 0.989 2.075 2.055 2.043 1.606 1.3771.139 98 97 57 36 14 2.14E−6 >1.00E−4 >1.00E−4 NCI-H23 0.496 1.650 1.4891.840 0.919 0.737 0.675 86 90 37 21 16 5.55E−7 >1.00E−4 >1.00E−4NCI-H322M 0.437 1.127 1.150 1.130 0.801 0.558 0.706 103 101 53 32 391.35E−6 >1.00E−4 >1.00E−4 NCI-H460 0.218 2.023 1.312 0.639 0.491 0.2720.285 61 23 15 3 3 1.93E−8 >1.00E−4 >1.00E−4 NCI-H522 0.818 1.811 1.7751.706 1.213 0.929 0.737 96 89 40 14 −10 6.22E−7 3.88E−5 >1.00E−4 ColonCancer COLO 205 0.239 0.893 0.884 0.804 0.506 0.310 0.308 99 86 41 11 116.38E−7 >1.00E−4 >1.00E−4 HCT-116 0.217 1.954 1.728 0.823 0.441 0.4480.308 87 23 13 13 6 3.81E−8 >1.00E−4 >1.00E−4 HCT-15 0.242 1.070 1.0230.967 0.479 0.273 0.148 94 68 29 4 −39 4.34E−7 1.22E−5 >1.00E−4 HT290.206 1.369 1.303 0.773 0.445 0.287 0.226 94 49 21 7 29.39E−8 >1.00E−4 >1.00E−4 KM12 0.448 1.635 1.610 1.365 0.740 0.536 0.50298 77 25 7 5 3.30E−7 >1.00E−4 >1.00E−4 SW-620 0.222 1.201 1.128 0.6070.390 0.320 0.321 92 39 15 10 10 6.30E−8 >1.00E−4 >1.00E−4 CNS CancerSF-258 0.374 1.402 1.439 0.850 0.547 0.573 0.592 104 45 27 19 218.62E−8 >1.00E−4 >1.00E−4 SF-295 0.589 1.716 1.768 1.661 0.959 0.7950.762 105 95 34 20 17 5.47E−7 >1.00E−4 >1.00E−4 SF-539 0.432 1.548 1.5561.290 0.774 0.659 0.460 101 77 31 20 2 3.81E−7 >1.00E−4 >1.00E−4 SNB-190.290 1.079 1.079 0.694 0.508 0.425 0.337 100 51 28 17 61.13E−7 >1.00E−4 >1.00E−4 SNB-75 0.615 1.216 1.187 1.169 1.039 0.8630.555 95 92 71 41 −10 5.03E−6 6.44E−5 >1.00E−4 U251 0.283 1.258 1.2720.864 0.570 0.473 0.266 98 53 29 19 −6 1.84E−7 5.66E−5 >1.00E−4 MelanomaLOXIMVI 0.265 1.475 1.547 1.266 0.461 0.404 0.450 106 52 16 11 153.08E−7 >1.00E−4 >1.00E−4 MALME-3M 0.534 1.015 0.997 0.999 1.043 0.8440.901 96 97 106 64 78 >1.00E−4 >1.00E−4 >1.00E−4 M14 0.388 1.627 1.6551.423 0.767 0.853 0.810 102 84 31 21 34 4.31E−7 >1.00E−4 >1.00E−4SK-MEL-2 0.891 2.450 2.421 2.364 1.428 1.298 1.156 98 95 35 27 165.58E−7 >1.00E−4 >1.00E−4 SK-MEL-23 0.578 1.603 1.650 1.574 1.130 0.9101.002 105 97 54 32 41 1.51E−8 >1.00E−4 >1.00E−4 SK-MEL-5 0.339 1.4141.355 1.270 0.716 0.460 0.167 95 87 35 11 −45 5.13E−7 1.59E−5 >1.00E−4UACC-257 0.609 1.981 2.013 1.239 1.063 0.692 0.686 102 46 33 21 66.45E−8 >1.00E−4 >1.00E−4 UACC-62 0.698 2.085 2.074 2.063 1.230 1.0751.139 99 99 38 27 32 6.41E−7 >1.00E−4 >1.00E−4 Ovarian Cancer IGROV10.152 0.529 0.502 0.422 0.244 0.173 0.194 93 72 24 6 112.87E−7 >1.00E−4 >1.00E−4 OVCAR-3 0.329 1.222 1.145 1.093 0.936 0.5250.457 91 86 66 22 14 2.48E−6 >1.00E−4 >1.00E−4 OVCAR-4 0.562 1.494 1.3361.345 1.330 0.843 0.738 83 84 82 30 19 4.16E−6 >1.00E−4 >1.00E−4 OVCAR-50.460 0.939 0.915 0.825 0.734 0.652 0.738 96 77 56 4758 >1.00E−4 >1.00E−4 OVCAR-6 0.323 1.364 1.390 0.821 0.689 0.567 0.508102 57 36 25 18 2.16E−7 >1.00E−4 >1.00E−4 SK-OV-3 0.341 1.046 1.0851.026 0.741 0.593 0.456 106 97 57 36 16 2.09E−6 >1.00E−4 >1.00E−4 RenalCancer 785-0 0.739 2.801 2.852 2.733 1.379 1.267 1.070 102 97 31 26 165.14E−7 >1.00E−4 >1.00E−4 A498 0.248 1.550 1.556 1.496 1.391 1.033 0.766101 91 74 14 −17 2.49E−6 2.85E−5 >1.00E−4 ACHN 0.459 1.484 1.472 1.3110.676 0.495 0.359 89 83 21 4 −22 3.42E−7 1.41E−5 >1.00E−4 CAKI-1 0.2581.038 1.078 1.014 0.670 0.472 0.397 105 97 51 25 151.08E−6 >1.00E−4 >1.00E−4 RXF 399 0.409 0.699 0.721 0.716 0.672 0.5700.351 108 107 91 56 −14 1.20E−5 6.26E−5 >1.00E−4 SN12C 0.345 1.182 1.1960.606 0.658 0.552 0.419 102 55 37 25 9 1.89E−7 >1.00E−4 >1.00E−4 TK-100.455 0.954 1.038 1.052 0.762 0.635 0.595 110 113 58 34 272.15E−6 >1.00E−4 >1.00E−4 UO-31 0.234 1.020 1.057 0.923 0.593 0.4810.476 105 88 46 31 31 7.90E−7 >1.00E−4 >1.00E−4 Prostate Cancer PC-30.499 1.471 1.514 1.577 0.384 0.675 0.489 104 111 40 18 −2 7.15E−77.99E−5 >1.00E−4 DU-145 0.195 0.694 0.857 0.589 0.453 0.320 0.243 95 5637 18 7 2.12E−7 >1.00E−4 >1.00E−4 Breast Cancer MCF7 0.227 1.235 1.0900.680 0.398 0.273 0.169 63 45 17 5 −26 7.34E−8 1.41E−5 >1.00E−4NCWADR-RES 0.646 1.868 1.863 1.863 1.187 0.998 0.942 100 100 44 29 247.87E−7 >1.00E−4 >1.00E−4 MDA-MB-231/ATCC 0.540 1.381 1.311 1.183 0.9190.781 0.793 92 70 45 29 30 6.41E−7 >1.00E−4 >1.00E−4 HS 576T 0.718 1.2261.248 1.212 1.077 0.774 0.628 104 97 71 11 −13 2.22E−6 2.94E−5 >1.00E−4MDA-MB-435 0.397 1.528 1.569 1.576 0.750 0.627 0.785 104 104 31 20 335.54E−7 >1.00E−4 >1.00E−4 BT-549 0.318 0.810 0.623 0.740 0.638 0.4750.304 103 85 65 32 −4 2.83E−6 7.58E−5 >1.00E−4 T-47D 0.387 0.901 0.8390.615 0.713 0.684 0.524 88 83 63 34 27 2.90E−6 >1.00E−4 >1.00E−4

Compound 3 underwent evaluation in a B16-F10 tumor model in vivo. Theantitumor activity of Compound 3 is shown in FIG. 7. When 25 mg/kg ofCompound 3 was administered to athymic mice three times weekly, itdemonstrated potent antitumor activity, with respect to tumor volume(FIG. 7A) and tumor growth rate (FIG. 7B) compared to administration ofmethotrexate and untreated animals (sham).

FIG. 8 shows the decrease in metastasis compared to the sham. FIG. 9shows the decrease in vascularity of the B 16-F10 tumors compared to thesham.

The results demonstrate compounds of structure 3 have antitumor activityin vitro and in vivo along with both cytostatic and cytotoxic activity.

Example 2 Compounds Derived from Compound of Formula II

An example of a compound derived from Compound II is as follows:

Eight compounds having different substituents for R were synthesized andtheir activity was assessed in various assays. The compounds, assays andthe results of these assays are shown in Table 5. A preferred compoundwas AAG145-344, in which R=H. This compound had a Flk-1 kinase activityof 5.1, which is lower than the Flk-1 kinase activity of 12.9 found forthe standard, SU5416.

TABLE 5 EGFR IRREVERSIBLE Flk-1 Flt-1 PDGFR- A431 CAM Compound kinaseEGFR kinase kinase β kinase cytotoxicity angiogenesis AAG145- 45.1 3.92335 R = 3-Cl AAG155- 98.8 167.5 20.30 336 R = 3-Br AAG145- >300 132.92.60 340 R = 4-Cl AAG145- >300 163.1 4.70 341 R = 4-Br AAG145- 23 198.5342 R = 2- isopropyl AAG145- 232.7 5.1 344 R = H AAG145- >300 73.0 346 R= 3-F AAG145- >300 21.9 347 R = 4-F SU5416 12.9 0.04 PD153035 0.23PD168393 0.13 0.521 cisplatin 10.6 DMBI 3.75 VEGF 11.9 inhibitor

Example 3 Synthesis of 5-Substituted ThiophenylPyrimido[4,5-b]indol-2,4-diamines

Chemical Discussion

The synthesis of target compounds commenced from commercially available1,2-dichloro-3-nitro-benzene 3 and ethyl cyanoacetate. Ethylcyanoacetate was initially treated with base potassium tert-butoxide and3 was added later to the reaction. Displacement of the chloro group of 3by the ethyl cyanoacetate anion provided compound 4 as a viscous yellowliquid. Reduction of the nitro group of 4 followed by autocyclizationproduced compound 5 as a pink solid. Use of fresh or activated zincpowder is recommended for this reaction. Cyclocondensation of 5 withcarbamimidic chloride hydrochloride 7 produced the tricyclic compound 8as a brown solid (synthesis of 7 is provided in Scheme 2). Protection ofthe 2-amino group of 8 using 2,2-dimethyl propanoic anhydride, underbasic conditions, produced 9. Compound 10 was prepared by treating 9with phosphoric trichloride at reflux. Displacement of the 4-chloro withammonia, and simultaneous deprotection of the 2-amino group of 10, wasachieved using a sealed vessel; thus the 2,4-diamino compound 11 wasobtained in 39% yield as a key intermediate. Treatment of 11 with theappropriate substituted benzenethiol provided target compounds 1 and 2,in yields of 97% and 87%, respectively. This reaction was carried outunder basic conditions in a microwave (Initiator® from Biotage).

Methods

All evaporations were carried out in vacuo with a rotary evaporator.Analytical samples were dried in vacuo in a Chem Dry drying apparatusover P₂O₅. Thin-layer chromatography (TLC) was performed on silica gelplates. Spots were visualized by UV light (254 and 365 nm). Purificationby column chromatography was carried out using Merck silica gel 60(200-400 mesh). The weight of silica gel for column chromatography wasin the range of 100-200 times the weight of the crude compound beingpurified. All columns were wet packed. Solvent systems were reportedratios of solvents. Melting points were determined on a MeI-Temp IImelting point apparatus with a digital thermometer and were uncorrected.¹H NMR spectra were recorded on a Bruker WH-300 (300 MHz) NMRspectrometer. The chemical shift (6) values are reported as parts permillion (ppm) relative to tetramethylsilane as internal standard;s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, bs=broadsinglet, exch=protons exchangeable by addition of D₂O. Elementalanalyses were performed by Atlantic Microlab. Inc., Norcross, Ga.Elemental compositions were +0.4% of the calculated values. All solventsand chemicals were purchased from Aldrich Chemical Co. and FisherScientific and were used as received.

Ethyl(2-chloro-2-nitrophenyl)(cyano)acetate (4)

To an ice cold solution of ethylcyanoacetate (10.9 mL, 102.4 mmol) inanhydrous THF (170 mL) under nitrogen, was added potassium tert-butoxide(12.7 g, 107.5 mmol). The formed white suspension was stirred for 15minutes, then treated with 2,3-dichloronitrobenzene (9.83 g, 51.2 mmol).The suspension was heated at reflux for 48 hours. The resulting reddishbrown solution was poured into water, and the aqueous mixture wasacidified to pH 2 with concentrated HCl. The mixture was extracted withether (3×150 mL) and then the combined organic phase was dried (usingNa₂SO₄) and concentrated to give a dark oil. Flash chromatography using10:1 hexane:ethyl acetate in a column packed with silica gel, 10 timesthe weight of the dark oil, provided a viscous yellow liquid 4, whichwas used without further purification for the next step. TLC Rf 0.23(hexane-ethyl acetate 3:1). ¹H NMR δ 1.33-1.38 (t, 3H, CH₃); 4.29-4.35(q, 2H, CH₂); 7.59-8.14 (m, 3H, phenyl).

Ethyl-2-amino-4-chloro-1H-indole-3-carboxylate (5)

4 (18 g, 67 mmol), in 250 mL glacial acetic acid, was treated with asingle charge of 18 g of zinc dust. The mixture was heated at 55° C. for45 minutes. Later, 6 g more zinc dust was added. After heating foranother 105 minutes, the yellow mixture was filtered through a pad ofcelite. The pad was washed with acetic acid and the filtrate wasconcentrated to a residue that was distributed between chloroform andwater. The organic phase was washed with NaHCO₃ (5%) to provide a pinkprecipitate which was filtered, dried over P₂O₅, dissolved in methanol,added silica gel and converted to a silica gel plug by removing thesolvent under reduced pressure. The plug was transferred on top of acolumn packed with silica gel, ten times the weight of plug, eluted withhexane, chloroform, 5% ethylacetate in chloroform and 10% ethylacetatein chloroform. Fractions containing the product 5 (TLC) were pooled andevaporated to give a pink solid. The overall yield from 3 to 5 was 63%.TLC Rf 0.187 (hexane-chloroform 1:1); mp 140-142° C.; ¹H NMR (DMSO-d6) δ1.25-1.28 (t, 3H, CH₃); 4.18-4.20 (q, 2H, CH₂); 6.85 (bs, 2H, 2-NH₂,exch); 6.92-7.09 (m, 3H, phenyl); 10.93 (bs, 1H, 9-NH, exch). Anal.Calculated (C₁₁H₁₁ClN₂O₂): C, 55.36; H, 4.65; N, 11.74; Cl, 14.85.Found: C, 55.39; H, 4.60; N, 11.65; Cl, 14.96.

Carbamimidic Chloride Hydrochloride (7).

Cyanamide (4.2 g, 0.1 mol) was dissolved in 100 mL of diethyl ether in a500 mL round bottom flask. The mixture was stirred under nitrogen. 100mL of 2M HCl in diethyl ether was added to the reaction flask via a 250mL dropping funnel. Stirring was continued for 2 hours at roomtemperature. The white salt which precipitated out was filtered anddried. The overall yield for 7 was 96%. 7 was used for the next stepwithout further purification.

2-Amino-5-chloro-3,9-dihydro-4H-pyrimido[4,5-b]indol-4-one (8)

1 g methyl sulfone was heated to melting. 7 (106.22 mg, 1.37 mmol) wasadded and the resulting mixture was stirred and heated at 110-120° C. todissolve completely. 5 (200 mg, 0.837 mmol) was added in one part to thereaction mixture. Stirring was continued for 30 minutes. About 10 mLwater was added to quench the reaction. Ammonia water was added toneutralize the reaction mixture. Solid precipitated out. This solid wasfiltered. Obtained solid was dissolved in chloroform and methanol, dried(using Na₂SO₄) and recrystallized. The overall yield was 78%. TLC Rf0.33 (chloroform-methanol 1:1); mp >250° C.; ¹H NMR (DMSO-d6) δ 6.57(bs, 2H, 2-NH₂, exch); 7.04-7.17 (m, 3H, phenyl); 10.41 (s, 1H, 9-NH,exch); 11.64 (s, 1H, 3-NH, exch). Anal. Calculated (C₁₀H₇ClN₄O.0.3CH₃OH): C, 50.65; H, 3.38; N, 22.94; Cl, 14.52. Found: C, 50.91; H,3.34; N, 22.60; Cl, 14.77.

N-(5-chloro-4-oxo-4,9-dihydro-3H-pyrimido[4,5-b]indol-2-yl)-2,2-dimethylpropanamide (9)

Compound 8 (300 mg, 1.27 mmol), 2-dimethyl propanoic anhydride (713.32mg, 3.83 mmol), dimethyl aminopyridine (7 mg, 0.06 mmol), triethylamine(514.05 mg, 5.08 mmol) were weighed together in a 50 mL round bottomflask. This flask was placed in an oil bath at 60° C. with stirring for2 days. Then, to the reaction mixture was added 1 g silica gel. The DMFwas removed using oil pump and a silica gel plug was made. The plug wastransferred on top of a column packed with silica gel, twenty times theweight of plug, eluted with chloroform, 1% methanol in chloroform and 5%methanol in chloroform. Fractions containing the product 9 (TLC) werepooled and evaporated to give solid compound. The overall yield was 40%.TLC Rf 0.45 (chloroform-methanol 10:1), m.p. 185.8-190.1° C., ¹H NMR: δ1.27 (s, 9H, pivaloyl); 7.19-7.40 (m, 3H, phenyl); 11.15 (s, 1H, 9-NH,exch); 11.94 (s, 1H, 9-NH, exch); 12.12 (s, 1H, 3-NH, exch).

N-(4,5-dichloro-9H-pyrimido[4,5-b]indol-2-yl)-2,2-dimethyl propanamide(10)

To 9 (2 g, 6.274 mmol) was added 30 mL of POCl₃ in a 250 mL round bottomflask. The reaction mixture was refluxed at 110-120° C. for 4 hours.After this the POCl₃ was evaporated and the mixture was neutralizedusing NH₄OH. The aqueous mixture was filtered (the precipitate being thecompound). The filtrate also contained some compound. Therefore, it wasextracted using chloroform and ethyl acetate. The precipitate obtainedwas dissolved in chloroform and methanol. Both the dissolved precipitateand extracted filtrate were dried using sodium sulfate overnight. To thesolution was added silica, and solvent was removed under reducedpressure to provide a silica gel plug. The plug was transferred on topof a column packed with silica gel, twenty times the weight of plug,eluted with chloroform, 1% methanol in chloroform and 5% methanol inchloroform. Fractions containing the product 10 (TLC) were pooled andevaporated to give a solid. The overall yield was 70%. TLC Rf 0.86(chloroform-methanol 5:1), m.p. 245.6-246.1° C., ¹H NMR: δ 1.24 (s, 9H,pivaloyl); 7.37-7.63 (m, 3H, phenyl); 10.32 (s, 1H, 9-NH, exch); 12.96(s, 1H, 2-NH, exch).

5-Chloro-9H-pyrimido[4,5-b]indole-2,4 diamine propanamide (11)

5 mL methanol was saturated with ammonia in the plastic container (ofthe sealed tube reaction apparatus), which was cooled in dry ice andacetone. 10 (200 mg, 0.6 mmol) was added to this methanol saturated withammonia. The solution was stirred at 130° C. for 2 days. Silica gel wasadded to the reaction mixture and methanol was removed under reducedpressure to make a plug. The plug was transferred on top of a columnpacked with silica gel, ten times the weight of plug, eluted withchloroform and 1% methanol in chloroform. Fractions containing theproduct 10 (TLC) were pooled and evaporated to give a solid. The overallyield was 39%. TLC Rf 0.43 (chloroform-methanol 5:1), m.p. 245.2-246.3°C., ¹H NMR: δ 6.15 (bs, 2H, 4-NH₂, exch); 6.85 (bs, 2H, 2-NH₂, exch),7.03-7.24 (m, 3H, phenyl); 11.54 (bs, 1H, 9-NH, exch). Anal. Calculated(C₁₀H₈ClN₅): C, 51.40; H, 3.38; N, 29.97; Cl, 15.17. Found: C, 51.58; H,3.46; N, 29.86; Cl, 15.05.

General procedure for the synthesis of5-(substituted-phenylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines 1 and 2

Compound 11 (50 mg, 0.2 mmol), the appropriate thiol (0.9 mmol), andpotassium carbonate (120 mg, 0.9 mmol) were added to a 2-5 mL capacitymicrowave vial. 3 mL NMP was added as solvent and the tube was sealed.The reaction was run in a microwave for 30 minutes at 250° C. Afterbeing cooled to room temperature, the reaction mixture was transferredon top of a 3 cm diameter column packed with approximately 40 cm silicagel and eluted with chloroform, 1% methanol in chloroform, 3% methanolin chloroform and 4% methanol in chloroform. Fractions containing theproduct (TLC) were pooled and evaporated to afford the product 1 and 2.

5-(phenylthio)-9H-pyrimido[4,5-b]indole-2,4-diamine (1)

Using the general procedure described above, compound 1 was obtained bythe reaction of 11 with benzene thiol to afford an off-white solid in97% yield. TLC Rf 0.23 (chloroform-methanol 10:1), m.p. >250° C., ¹HNMR: δ 6.03 (bs, 2H, 4-NH₂, exch); 7.01-7.41 (m, 10H, Ar—H×8, 2-NH₂);11.48 (bs, 1H, 9-NH, exch). Anal. Calculated (C₁₆H₁₃N₅S): C, 62.52; H,4.26; N, 22.78; S, 10.43. Found: C, 62.51; H, 4.51; N, 22.44; S, 10.35.

5-[(4-methylphenyl)thio]-9H-pyrimido[4,5-b]indole-2,4-diamine (2)

Using the general procedure described above, compound 2 was obtained bythe reaction of 11 with 4-methylbenzene thiol to afford an off-whitesolid in 87% yield. TLC Rf 0.54 (chloroform-methanol 5:1), m.p. >250°C., ¹H NMR: δ 2.19 (s, 3H, CH₃), 6.02 (bs, 2H, 4-NH₂, exch); 7.19 (bs,2H, 2-NH₂, exch), 6.97-7.36 (m, 7H, Ar—H×7); 11.46 (bs, 1H, 9-NH, exch).Anal. Calculated (C₁₇H₁₅N₅S): C, 63.53; H, 4.70; N, 21.70; S, 9.98.Found: C, 62.39; H, 4.56; N, 21.00; S, 9.56.

Chemical Discussion

The synthesis route used to prepare analogues is described in Scheme 3.(2-ethoxycarbonylmethyl-phenyl)-acetic acid ethyl ester 2 (71%) wasobtained by hydrolysis of 1,2-phenylenediacetonitrile 1 withconcentrated H₂SO₄. Compound 2 with sodium ethoxide in ethanol gavecompound 3 (61%), which further reacted with guanidine in the presenceof potassium t-butoxide under 150° C. in a microwave reaction to givecompound 4 (34%). To access target compounds, first, compound 4 wastried to turn the 4-oxo to 4-chloro, which traditionally is adopted insynthesizing 4-anilino substituted analogues. However, because of thelower aromaticity of the ring, the chlorination did not run well underknown reaction conditions. Compound 4 was decomposed when refluxed withPOCl₃. Thus, the 4-oxo was converted to 4-sulfonate, which is a goodleaving group for SNi reaction. Compound 5 was obtained when treatingcompound 4 with the 4-nitrobenzensulfonyl chloride (36%). The targetcompound 6 with different substitution on the phenyl ring was obtainedwhen refluxing compound 5 with corresponding aniline for 12-18 h.

Methods (2-Ethoxycarbonylmethyl-phenyl)-acetic acid ethyl ester (2)

1,2-phenylenediacetonitrile (1.0 g, 6.40 mmol) was dissolved in 5 mlethyl alcohol and 2 ml concentrated sulfuric acid in a 25 ml roundbottom flask. The mixture was stirred and heated to reflux for 6 hours.After neutralizing the reaction solution with ammonium hydroxide, theresult solution was extracted with ethyl acetate (3×50 ml). The organicphase was combined and dried with Na₂SO₄. Concentration of the ethylacetate afforded a yellow liquid. Running column with hexane: acetylacetate=10:1, got yellow liquid 1.13 g (70.6%). ¹H NMR (DMSO-d6): δ 1.24(t, 6H, CH₃), δ 3.7 (s, 4H, CH₂CO), δ 4.16 (q, 4H, OCH₂), δ 7.3 (d, 4H,Ar—H).

2-oxo-indan-1-carboxylic acid ethyl (3)

Compound 2 (5.6 g, 2 mmol) was diluted in 30 ml ethyl alcohol, and 1.6 g(2.2 mmol) sodium ethoxide was slowly added in a 150 ml round bottomflask, stirring at room temperature for 4 hours to form a yellow clearsolution. After neutralizing the reaction solution with dilutehydrochloric acid, the result solution was extracted with ethyl acetate(3×50 ml). The organic phase was combined and dried with Na₂SO₄.Concentration of the ethyl acetate afforded a brown solid. Runningcolumn with hexane: acetyl acetate=10:1, got 2.80 g (61%) white solid.M.p: 59˜61° C. ¹H NMR (DMSO-d6): δ 3.64 (s, 2H, Ar—CH₂CO), δ 4.43 (q,2H, OCH₂), δ 7.6 (m, 4H, Ar—H), δ 11.0 (s, 1H, OH, exch).

2-amino-3,9-dihydro-indeno[2,1-d]pyrimidin-4-one (4)

Compound 3 (0.1 g, 0.49 mmol), guanidine hydrochloride (0.05 g, 0.52mmol) and potassium t-butoxide (0.12 g, 1.1 mmol) were dissolved in 5 mlt-butanol. The condition of microwave reaction is 140° C., 3 hours. Thesolid was filtered out and washed with methanol. The filtrate wascombined and plugs were made with silica gel. Running column withchloroform:methanol=10:1 afforded light yellow solid 23 mg (34%). Mp:˜330° C. (dec.). ¹H NMR (DMSO-d6): δ 3.65 (s, 2H, CH₂), δ 6.71 (s, 2H,NH₂, exch), δ 7.0˜7.67 (m, 4H, Ar—H), δ 10.93 (s, 1H, NH, exch). CHNAnal. (C₁₁H₉N₃O.0.1H₂O): C, H, N.

4-Nitro-benesulfonic acid 2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester(5)

A solution of 4 (0.3 g, 1.5 mmol), triethylamine (0.42 ml, 3 mmol), DMAP(20 mg) and 4-nitrobenzenesulfonyl chloride (0.67 g, 3 mmol) indichloromethane 40 ml was stirred at room temperature for 4 hours. Tothe reaction solution was added 1.5 g silica gels to make plugsdirectly. The column was eluted with Hexane: Chloroform (2:1). Fractionscontaining the product were pooled and evaporated to afford purecompound 10 as yellow solid 0.21 g (36%). TLC Rf 0.47 (CHCl₃/CH₃OH,10:1). Mp: 189.6° C. (dec); ¹H NMR (DMSO-d6): δ 3.92 (s, 2H, CH₂), δ7.18 (br, 2H, NH₂, exch), δ 7.15-7.6 (m, 4H, Ph-H), δ 8.4-8.6 (m, 4H,4-NO₂-Ph-H). HRMS (EI): calculation for C₁₇H₁₃N₄O₅S 385.0607, found,385.0584.

General Procedure for the Synthesis of Compounds 7-19.

A 50 ml round-bottom flask was charged with compound 5, substitutedthiophenol or aniline (molar ration 1:2) and anhydrous 1,4-dioxane 10ml. The mixture was heated and kept refluxing for 12-18 hours. Plugswere made directly after stopping the reaction. The column was elutedwith hexane/chloroform (2:1). Fractions containing the product werepooled and evaporated to afford pure compound.

N⁴-(2-Isopropyl-phenyl)-9H-indeno[2,1-d]pyrimidine-2,4-diamine (7)

Compound was synthesized from 4-nitro-benesulfonic acid2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester 5 (0.1 g, 0.26 mmol) usingthe general procedure described above to afford 15 mg (18%) as a lightbrown solid. TLC Rf 0.43 (Et₃N/EtOAc/Hex, 1:3:5). Mp: 193.4˜195.3° C.;¹H NMR (DMSO-d6): δ 1.14-1.16 (d, 6H, 2CH₃), δ 3.10-3.24 (m, H, CH), δ3.70 (s, 2H, CH₂), δ 6.13 (br, 2H, NH₂, exch), δ 7.11-7.93 (m, 8H,Ph-H), δ 7.91 (s, H, NH, exch). HRMS (EI): calculation for C₂₀H₂₁N₄,317.1766, found, 317.1751.

N⁴-(4-Isopropyl-phenyl)-9H-indeno[2,1-d]pyrimidine-2,4-diamine (8)

Compound was synthesized from 4-nitro-benesulfonic acid2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester 5 (75 mg, 0.76 mmol) usingthe general procedure described above to afford 36.4 mg (59%) as a lightbrown solid. TLC Rf 0.30 (CHCl₃/CH₃OH, 10:1). Mp: 178.5˜180.4° C.; ¹HNMR (DMSO-d6): δ 3.76 (s, 2H, CH₂), δ 6.5 (br, 2H, NH₂, exch), δ7.14-7.92 (m, 8H, Ph-H), δ 8.33 (s, H, NH, exch). HRMS (EI): calculationfor C₂₀H₂₀N₄, 316.1688, found, 316.1704.

N⁴-(3-Fluoro-phenyl)-9H-indeno[2,1-d]pyrimidine-2,4-diamine (9)

Compound was synthesized from 4-nitro-benesulfonic acid2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester 5 (0.15 g, 0.39 mmol) usingthe general procedure described above to afford 15 mg (13%) as a lightbrown solid. TLC Rf 0.42 (CHCl₃/CH₃OH, 10:1). Mp: 198.7˜200.1° C.; ¹HNMR (DMSO-d6): δ 3.8 (s, 2H, CH₂), δ 6.6 (br, 2H, NH₂, exch), δ 7.1-7.9(m, 8H, Ph-H), δ 8.4 (s, H, NH, exch). HRMS (EI): calculation forC₁₇H₁₃FN₄, 292.1124, found, 292.1123.

N⁴-(3-Chloro-phenyl)-9H-indeno[2,1-d]pyrimidine-2,4-diamine (10)

Compound was synthesized from 4-nitro-benesulfonic acid2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester 5 (0.1 g, 0.26 mmol) usingthe general procedure described above to afford 22.8 mg (35%) as a lightbrown solid. TLC Rf 0.65 (CHCl₃/CH₃OH, 10:1). Mp: 224.1˜225.2° C.; ¹HNMR (DMSO-d6): δ 3.7 (s, 2H, CH₂), δ 6.5 (br, 2H, NH₂, exch), δ 7.0-7.8(m, 8H, Ph-H), δ 8.3 (s, H, NH, exch). HRMS (EI): calculation forC₁₇H₁₃CN₄, 308.0829, found, 308.0838.

N⁴-(3-Bromo-phenyl)-9H-indeno[2,1-d]pyrimidine-2,4-diamine (11)

Compound was synthesized from 4-nitro-benesulfonic acid2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester 5 (0.1 g, 0.26 mmol) usingthe general procedure described above to afford 46 mg (50%) as a lightbrown solid. TLC Rf 0.47 (Et₃N/EtOAc/Hex, 1:3:5). Mp: 233-236° C.; ¹HNMR (DMSO-d6): δ 3.76 (s, 2H, CH₂), δ 6.5 (br, 2H, NH₂, exch), δ7.14-7.92 (m, 8H, Ph-H), δ 8.33 (s, H, NH, exch). HRMS (EI): calculationfor C₁₇H₁₄BrN₄, 353.0402, found, 353.0387.

N⁴-(4-Fluoro-phenyl)-9H-indeno[2,1-d]pyrimidine-2,4-diamine (12)

Compound was synthesized from 4-nitro-benesulfonic acid2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester 5 (0.12 g, 0.31 mmol) usingthe general procedure described above to afford 46 mg (50%) as a lightbrown solid. TLC Rf 0.54 (CHCl₃/CH₃OH, 10:1). Mp: 204.7˜205.8° C.; ¹HNMR (DMSO-d6): δ 3.72 (s, 2H, CH₂), δ 6.38 (br, 2H, NH₂, exch), δ7.11-7.89 (m, 8H, Ph-H), δ 8.19 (s, H, NH, exch). CHN Anal(C₁₇H₁₃FN₄.0.4H₂O): C, H, N, F.

N⁴-(4-Chloro-phenyl)-9H-indeno[2,1-d]pyrimidine-2,4-diamine (13)

Compound was synthesized from 4-nitro-benesulfonic acid2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester 5 (0.10 g, 0.26 mmol) usingthe general procedure described above to afford 30 mg (38%) as a lightbrown solid. TLC Rf 0.33 (CHCl₃/CH₃OH, 10:1). Mp: 215.9˜216.8° C.; ¹HNMR (DMSO-d6): δ 1.16-1.22 (d, 6H, 2CH₃), δ 2.6-2.9 (m, H, CH), δ 3.7(s, 2H, CH₂), δ 6.3 (br, 2H, NH₂, exch), δ 7.2-7.8 (m, 8H, Ph-H), δ 8.1(s, H, NH, exch). CHN Anal (C₁₇H₁₃ClN₄): C, H, N, Cl.

N⁴-(4-Bromo-phenyl)-9H-indeno[2,1-d]pyrimidine-2,4-diamine (14)

Compound was synthesized from 4-nitro-benesulfonic acid2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester 5 (0.13 g, 0.34 mmol) usingthe general procedure described above to afford 72 mg (61%) as awhite-off solid. TLC Rf 0.44 (CHCl₃/CH₃OH, 10:1). Mp: 213.6˜214.8° C.;¹H NMR (DMSO-d6): δ 3.75 (s, 2H, CH₂), δ 6.45 (br, 2H, NH₂, exch), δ7.14-7.89 (m, 8H, Ph-H), δ 8.31 (s, H, NH, exch). CHN Anal(Ct₇H₁₃BrN₄.0.6CH₃OH): C, H, N, Br.

N⁴-(4-Trifluoromethyl-phenyl)-9H-indeno[2,1-d]pyrimidine-2,4-diamine(15)

Compound was synthesized from 4-nitro-benesulfonic acid2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester 5 (0.22 g, 0.57 mmol) usingthe general procedure described above to afford 44 mg (27%) as a lightbrown solid. TLC Rf 0.48 (CHCl₃/CH₃OH, 10:1). Mp: 229.2˜230.8° C.; ¹HNMR (DMSO-d6): δ 3.8 (s, 2H, CH₂), δ 6.6 (br, 2H, NH₂, exch), δ 7.2-8.0(m, 8H, Ph-H), δ 8.7 (s, H, NH, exch). CHN Anal (C₁₈H₁₃F₃N₄): C, H, N,F.

N⁴-(3-Methoxyl-phenyl)-9H-indeno[2,1-d]pyrimidine-2,4-diamine (16)

Compound was synthesized from 4-nitro-benesulfonic acid2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester 5 (75 mg, 0.76 mmol) usingthe general procedure described above to afford 26.6 mg (45%) as a lightbrown solid. TLC Rf 0.47 (CHCl₃/CH₃OH, 10:1). Mp: 186.6-187.4° C.; ¹HNMR (DMSO-d6): δ 3.7 (s, 2H, CH₂), δ 3.8 (s, 3H, CH₃), δ 6.4 (br, 2H,NH₂, exch), δ 6.6-7.8 (m, 8H, Ph-H), δ 8.1 (s, H, NH, exch). HRMS (EI):calculation. for C₁₈H₁₆N₄O, 304.1324, found, 304.1390.

N⁴-Phenyl-9H-indeno[2,1-d]pyrimidine-2,4-diamine (17)

Compound was synthesized from 4-nitro-benesulfonic acid2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester 5 (0.05 g, 0.13 mmol) usingthe general procedure described above to afford 13.8 mg (37%) as a lightbrown solid. TLC Rf 0.37 (CHCl₃/CH₃OH, 10:1). Mp: 215.3-216.9° C.; ¹HNMR (DMSO-d6): δ 3.7 (s, 2H, CH₂), δ 6.4 (br, 2H, NH₂, exch), δ 7.0-7.9(m, 8H, Ph-H), δ 8.2 (s, H, NH, exch). HRMS (EI): calculation. forC₁₇H₁₄N₄, 274.1218, found, 274.1218.

N⁴-(4-Fluoro-3-chloro-phenyl)-9H-indeno[2,1-d]pyrimidine-2,4-diamine(18)

Compound was synthesized from 4-nitro-benesulfonic acid2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester 5 (0.1 g, 0.26 mmol) usingthe general procedure described above to afford 53 mg (52%) as a lightbrown solid. TLC Rf 0.43 (CHCl₃/CH₃OH, 10:1). Mp: 231.7-232.8° C.; ¹HNMR (DMSO-d6): δ 3.7 (s, 2H, CH₂), δ 6.5 (br, 2H, NH₂, exch), δ 7.1-8.0(m, 8H, Ph-H), δ 8.3 (s, H, NH, exch). HRMS (EI): calculation forC₁₇H₁₂ClFN₄, 326.0735, found, 326.0744.

N⁴-(2-Fluoro-4-chloro-phenyl)-9H-indeno[2,1-d]pyrimidine-2,4-diamine(19)

Compound was synthesized from 4-nitro-benesulfonic acid2-amino-9H-indeno[2,1-d]pyrimidin-4-ylester 5 (50 mg, 0.59 mmol) usingthe general procedure described above to afford 15 mg (18%) as a lightbrown solid. TLC Rf 0.58 (Et₃N/EtOAc/Hex, 1:3:5). Mp: 234.7-235.7° C.;¹H NMR (DMSO-d6): δ 3.98 (s, 2H, CH₂), δ 6.37 (br, 2H, NH₂, exch), δ7.15-7.86 (m, 7H, Ph-H), δ 8.16 (s, H, NH, exch). CHN Anal.(C₁₇H₁₂ClFN₄): C, H, N, Cl, F.

Elemental analysis for Compounds 4, 12-15 and 19 is shown in FIG. 10.

High-resolution mass spectra are shown in Table 6.

TABLE 6 High-Resolution mass spectra (HRMS) (EI) # Formula Calcd massFound mass 5 C₁₇H₁₃N₄O₅S 385.0607 385.0584 7 C₂₀H₂₁N₄ 317.1766 317.17518 C₂₀H₂₀N₄ 316.1688 316.1704 9 C₁₇H₁₃FN₄ 292.1124 292.1123 10 C₁₇H₁₃ClN₄308.0829 308.0838 11 C₁₇H₁₄BrN₄ 353.0402 353.0387 16 C₁₈H₁₆N₄O 304.1324304.1390 17 C₁₇H₁₄N₄ 274.1218 274.1218 18 C₁₇H₁₂ClFN₄ 326.0735 326.0744

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims.

1. A compound of formula I:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4b-8a, 5-6 and 7-8; the C ring may havean N or substituted N depending on the saturation level of the C ring,and the substitution may be all of R₁, R₂ and R₃; X and/or Y=N, NH, O,S, C; P═NR₄, O, S, CR₄R₅, wherein R₄ and R₅=lower alkyl, alkene, alkyne,and all of R₁ and R₂; R₁ and/or R₂=H, alkyl, a cycloalkyl having 6 orless carbons, alkene, alkyne, carbonyl, aryl, heteroaryl, substitutedaryl, substituted heteroaryl such as benzene, pyridine, biphenyl,bipyridine, quinazoline, isoquinoline, alkylaryl, alkylheteroaryl,substituted alkylaryl or alkylheteroaryl; Z=S, O, NR₅, CR₆R₇, S—C, C—S,O—CR₆, CR₆—O, NR₆—C, C—NR₆, CR₆—NR₇ or CR₆R₇, wherein R₅, R₆, R₇═H or alower alkyl, alkene or alkyne having 6 or less C atoms; wherein Z may beattached to the C ring at positions 5, 6, 7 or 8 and may be the same ordifferent and be attached to one or more positions on the ring; whereinZ may be zero and R₃ may be directly attached to the C-ring at positions5, 6, 7, and/or 8; wherein when the C-ring is saturated or partiallysaturated the substituted Z or R₃ creates chirality when P═C and R₆ andR₇ are different, then all stereoisomers thereof both separately and asracemic and/or diastereoisomeric mixtures are included; R₃=H, alkyl,cycloalkyl, aryl, heteroaryl, substituted aryl, substituted heteroaryl,alkylaryl, alkylheteroaryl and substituted saturated or unsaturatedalkylheteroaryl and alkylheterocyclic, alkylaryl, p-, m-,o-benzoyl-L-glutamate or 2,5-, 2,4-thienoyl-L-glutamate when the benzeneand thiophene ring may or may not have additional substitutions such asF, mono-, bi- and tricyclic aryl, heteroaryl or combinations thereof,ring substitutions such as biphenyl, bipyridyl or a phenyl-pyridyl orfused such as a quinoline or naphthyl including substituted systems suchas a 2-chloro,4-biphenyl and tricyclic and substituted tricyclicsystems.
 2. The compound according to claim 1, wherein Y=NH; R₂=H;P═NR₄; R₄=H; X=NH; R₁=H; Z=S and R₃=a cycloalkyl.
 3. The compoundaccording to claim 1, wherein Y=NH; R₂=H; P═CR₄R₅; R₄=H; R₅=H; X=NH;R₁=a substituted aryl; Z=0; and R₃=H.
 4. The compound according to claim3, wherein the substituted aryl is a chloride atom.
 5. A compound offormula II:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4b-8a, 5-6 and 7-8; the C ring may havean N or substituted N depending on the saturation level of the C ring,and the substitution may be all of R₁, R₂ and R₃; X and/or Y=N, NH, O,S, C; P═NR₄, O, S, CR₄R₅, wherein R₄ and R₅=lower alkyl, alkene, alkyne,and all of R₁ and R₂; R₁ and/or R₂=H, alkyl, a cycloalkyl having 6 orless carbons, alkene, alkyne, carbonyl, aryl, heteroaryl, substitutedaryl, substituted heteroaryl such as benzene, pyridine, biphenyl,bipyridine, quinazoline, isoquinoline, alkylaryl, alkylheteroaryl,substituted alkylaryl or alkylheteroaryl; Z=S, O, NR₅, CR₆R₇, S—C, C—S,O—CR₆, CRr—O, NR₆—C, C—NR₆, CRr—NR₇ or CR₆R₇, wherein R₅, R₆, R₇═H or alower alkyl, alkene or alkyne having 6 or less C atoms; wherein Z may beattached to the C ring at positions 5, 6, 7 or 8 and may be the same ordifferent and be attached to one or more positions on the ring; whereinZ may be zero and R₃ may be directly attached to the C-ring at positions5, 6, 7, and/or 8; wherein when the C-ring is saturated or partiallysaturated the substituted Z or R₃ creates chirality when P═C and R₆ andR₇ are different, then all stereoisomers thereof both separately and asracemic and/or diastereoisomeric mixtures are included; R₃=H, alkyl,cycloalkyl, aryl, heteroaryl, substituted aryl, substituted heteroaryl,alkylaryl, alkylheteroaryl and substituted saturated or unsaturatedalkylheteroaryl and alkylheterocyclic, alkylaryl, p-, m-,o-benzoyl-L-glutamate or 2,5-, 2,4-thienoyl-L-glutamate when the benzeneand thiophene ring may or may not have additional substitutions such asF, mono-, bi- and tricyclic aryl, heteroaryl or combinations thereof,ring substitutions such as biphenyl, bipyridyl or a phenyl-pyridyl orfused such as a quinoline or naphthyl including substituted systems suchas a 2-chloro,4-biphenyl and tricyclic and substituted tricyclicsystems.
 6. The compound according to claim 5, wherein Y=NH; R₂=H;P═CR₄R₅; R₄=H; R₅=H; X=NH; R₁=cycloalkyl; Z=0; and R₃=H.
 7. A method ofinhibiting receptor tyrosine kinase(s), dihydrofolate reductase,thymidylate synthase and/or dihydroorotate dehydrogenase activity in ananimal or human in need thereof, comprising administering to said animalor human a therapeutically effective amount in unit dosage form of acompound of formula I:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4b-8a, 5-6 and 7-8; the C ring may havean N or substituted N depending on the saturation level of the C ring,and the substitution may be all of R₁, R₂ and R₃; X and/or Y=N, NH, O,S, C; P═NR₄, O, S, CR₄R₅, wherein R₄ and R₅=lower alkyl, alkene, alkyne,and all of R₁ and R₂; R₁ and/or R₂=H, alkyl, a cycloalkyl having 6 orless carbons, alkene, alkyne, carbonyl, aryl, heteroaryl, substitutedaryl, substituted heteroaryl such as benzene, pyridine, biphenyl,bipyridine, quinazoline, isoquinoline, alkylaryl, alkylheteroaryl,substituted alkylaryl or alkylheteroaryl; Z=S, O, NR₅, CR₆R₇, S—C, C—S,O—CR₆, CR₆—O, NR₆—C, C—NR₆, CR₆—NR₇ or CR₆R₇, wherein R₅, R₆, R₇=H or alower alkyl, alkene or alkyne having 6 or less C atoms; wherein Z may beattached to the C ring at positions 5, 6, 7 or 8 and may be the same ordifferent and be attached to one or more positions on the ring; whereinZ may be zero and R₃ may be directly attached to the C-ring at positions5, 6, 7, and/or 8; wherein when the C-ring is saturated or partiallysaturated the substituted Z or R₃ creates chirality when P═C and R₆ andR₇ are different, then all stereoisomers thereof both separately and asracemic and/or diastereoisomeric mixtures are included; R₃=H, alkyl,cycloalkyl, aryl, heteroaryl, substituted aryl, substituted heteroaryl,alkylaryl, alkylheteroaryl and substituted saturated or unsaturatedalkylheteroaryl and alkylheterocyclic, alkylaryl, p-, m-,o-benzoyl-L-glutamate or 2,5-, 2,4-thienoyl-L-glutamate when the benzeneand thiophene ring may or may not have additional substitutions such asF, mono-, bi- and tricyclic aryl, heteroaryl or combinations thereof,ring substitutions such as biphenyl, bipyridyl or a phenyl-pyridyl orfused such as a quinoline or naphthyl including substituted systems suchas a 2-chloro,4-biphenyl and tricyclic and substituted tricyclicsystems.
 8. The method according to claim 7, wherein Y=NH; R₂=H; P═NR₄;R₄=H; X=NH; R₁=H; Z=S and R₃=a cycloalkyl.
 9. The method according toclaim 7, wherein Y=NH; R₂=H; P═CR₄R₅; R₄=H; R₅=H; X=NH; R₁=a substitutedaryl; Z=0; and R₃═H.
 10. The method according to claim 9, wherein thesubstituted aryl is a chloride atom.
 11. The method according to claim7, wherein the route of administration of the compound of formula I isparenteral, oral or intraperitoneal.
 12. The method according to claim11, wherein the parenteral route of administration is selected from thegroup consisting of intravenous; intramuscular; interstitial,intraarterial; subcutaneous; intraocular; intracranial;intraventricular; intrasynovial; transepithelial, including transdermal,pulmonary via inhalation, ophthalmic, sublingual and bucal; topical,including dermal, ocular, rectal and nasal inhalation via insufflationor nebulization.
 13. The method according to claim 7, wherein the unitdosage is administered orally in the form of hard or soft shell gelatincapsules, tablets, troches, sachets, lozenges, elixirs, suspensions,syrups, wafers, powders, granules, solutions or emulsions.
 14. Themethod according to claim 12, wherein the nasal administration of thecompound of formula I is selected from the group consisting of aerosols,atomizers and nebulizers.
 15. The method according to claim 7, whereinsaid animal or human is afflicted with a proliferative disease ordisorder selected from the group consisting of leukemia, non-small celllung cancer, colon cancer, central nervous system cancer, melanoma,ovarian cancer, renal cancer, prostate cancer, breast cancer; maculardegeneration and retinopathies.
 16. A method of inhibiting receptortyrosine kinase(s), dihydrofolate reductase, thymidylate synthase and/ordihydroorotate dehydrogenase activity in an animal or human in needthereof, comprising administering to said animal or human atherapeutically effective amount in unit dosage form of a compound offormula II:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4b-8a, 5-6 and 7-8; the C ring may havean N or substituted N depending on the saturation level of the C ring,and the substitution may be all of R₁, R₂ and R₃; X and/or Y=N, NH, O,S, C; P═NR₄, O, S, CR₄R₅, wherein R₄ and R₅=lower alkyl, alkene, alkyne,and all of R₁ and R₂; R₁ and/or R₂=H, alkyl, a cycloalkyl having 6 orless carbons, alkene, alkyne, carbonyl, aryl, heteroaryl, substitutedaryl, substituted heteroaryl such as benzene, pyridine, biphenyl,bipyridine, quinazoline, isoquinoline, alkylaryl, alkylheteroaryl,substituted alkylaryl or alkylheteroaryl; z=S, O, NR₅, CR₆R₇, S—C, C—S,O—CR₆, CR₆—O, NR₆—C, C—NR₆, CR₆—NR₇ or CR₆R₇, wherein R₅, R₆, R₇=H or alower alkyl, alkene or alkyne having 6 or less C atoms; wherein Z may beattached to the C ring at positions 5, 6, 7 or 8 and may be the same ordifferent and be attached to one or more positions on the ring; whereinZ may be zero and R₃ may be directly attached to the C-ring at positions5, 6, 7, and/or 8; wherein when the C-ring is saturated or partiallysaturated the substituted Z or R₃ creates chirality when P═C and R₆ andR₇ are different, then all stereoisomers thereof both separately and asracemic and/or diastereoisomeric mixtures are included; R₃=H, alkyl,cycloalkyl, aryl, heteroaryl, substituted aryl, substituted heteroaryl,alkylaryl, alkylheteroaryl and substituted saturated or unsaturatedalkylheteroaryl and alkylheterocyclic, alkylaryl, p-, m-,o-benzoyl-L-glutamate or 2,5-, 2,4-thienoyl-L-glutamate when the benzeneand thiophene ring may or may not have additional substitutions such asF, mono-, bi- and tricyclic aryl, heteroaryl or combinations thereof,ring substitutions such as biphenyl, bipyridyl or a phenyl-pyridyl orfused such as a quinoline or naphthyl including substituted systems suchas a 2-chloro,4-biphenyl and tricyclic and substituted tricyclicsystems.
 17. The method according to claim 16, wherein Y=NH; R₂=H;P═CR₄R₅; R₄=H; R₅=H; X=NH; R₁=cycloalkyl; Z=0; and R₃=H.
 18. The methodaccording to claim 16, wherein the route of administration of thecompound of formula II is parenteral, oral or intraperitoneal.
 19. Themethod according to claim 18, wherein the parenteral route ofadministration is selected from the group consisting of intravenous;intramuscular; interstitial, intraarterial; subcutaneous; intraocular;intracranial; intraventricular; intrasynovial; transepithelial,including transdermal, pulmonary via inhalation, ophthalmic, sublingualand buccal; topical, including dermal, ocular, rectal and nasalinhalation via insufflation or nebulization.
 20. The method according toclaim 19, wherein the nasal administration of the compound of formula IIis selected from the group consisting of aerosols, atomizers andnebulizers.
 21. The method according to claim 16, wherein the unitdosage is administered orally in the form of hard or soft shell gelatincapsules, tablets, troches, sachets, lozenges, elixirs, suspensions,syrups, wafers, powders, granules, solutions or emulsions.
 22. Themethod according to claim 16, wherein said animal or human is afflictedwith a proliferative disease or disorder selected from the groupconsisting of leukemia, non-small cell lung cancer, colon cancer,central nervous system cancer, melanoma, ovarian cancer, renal cancer,prostate cancer, breast cancer; macular degeneration and retinopathies.